Development of an Automated Morphometric Approach to Assess Vascular Outcomes following Exposure to Environmental Chemicals in Zebrafish.

Abstract

Background: Disruptions in vascular formation attributable to chemical insults is a pivotal risk factor or potential etiology of developmental defects and various disease settings. Among the thousands of chemicals threatening human health, the highly concerning groups prevalent in the environment and detected in biological monitoring in the general population ought to be prioritized because of their high exposure risks. However, the impacts of a large number of environmental chemicals on vasculature are far from understood. The angioarchitecture complexity and technical limitations make it challenging to analyze the entire vasculature efficiently and identify subtle changes through a high-throughput in vivo assay.

Objectives: We aimed to develop an automated morphometric approach for the vascular profile and assess the vascular morphology of health-concerning environmental chemicals.

Methods: High-resolution images of the entire vasculature in Tg(fli1a:eGFP) zebrafish were collected using a high-content imaging platform. We established a deep learning-based quantitative framework, ECA-ResXUnet, combined with MATLAB to segment the vascular networks and extract features. Vessel scores based on the rates of morphological changes were calculated to rank vascular toxicity. Potential biomarkers were identified by vessel-endothelium-gene-disease integrative analysis.

Results: Whole-trunk blood vessels and the cerebral vasculature in larvae exposed to 150 representative chemicals were automatically segmented as comparable to human-level accuracy, with sensitivity and specificity of 95.56% and 95.81%, respectively. Chemical treatments led to heterogeneous vascular patterns manifested by 31 architecture indexes, and the common cardinal vein (CCV) was the most affected vessel. The antipsychotic medicine haloperidol, flame retardant 2,2-bis(chloromethyl)trimethylenebis[bis(2-chloroethyl) phosphate], and tert-butylphenyl diphenyl phosphate ranked as the top three in vessel scores. Pesticides accounted for the largest group, with a vessel score of $\ge 1$, characterized by a remarkable inhibition of subintestinal venous plexus and delayed development of CCV. Multiple-concentration evaluation of nine per- and polyfluoroalkyl substances (PFAS) indicated a low-concentration effect on vascular impairment and a positive association between carbon chain length and benchmark concentration. Target vessel-directed single-cell RNA sequencing of $fli1a^{+}$ cells from larvae treated with $\lambda \text{-cyhalothrin}$, perfluorohexanesulfonic acid, or benzylbutyl phthalate, along with vessel-endothelium-gene-disease integrative analysis, uncovered potential associations with vascular disorders and identified biomarker candidates.

Discussion: This study provides a novel paradigm for phenotype-driven screenings of vascular-disrupting chemicals by converging morphological and transcriptomic profiles at a high-resolution level, serving as a powerful tool for large-scale toxicity tests. Our approach and the high-quality morphometric data facilitate the precise evaluation of vascular effects caused by environmental chemicals. https://doi.org/10.1289/EHP13214.